Isotope-coded ionization-enhancing reagents (ICIER) for high-throughput protein identification and quantitation using matrix-assisted laser desorption ionization mass spectrometry

ABSTRACT

Arginine-containing cysteine-modifying compounds useful for MALDI-MS analysis of proteins are provided. These compounds termed isotope-coded ionization enhancement reagents (ICIER) can provide ionization enhancement in MALDI-MS, relative quantitation, and additional database searching constraints at the same time without any extra sample manipulation. More specifically, ICIER increase the ionization efficiency of cysteine-containing peptides by attachment of a guanidino functional group. ICIER also increase the overall hydrophilicity of these peptides due to the hydrophilic nature of ICIER and thus increase the percentage of recovery of these peptides during sample handling and processing such as in-gel digestion or liquid chromatography. Finally, a combination of both light and heavy ICIER provides an accurate way to obtain relative quantitation of proteins by MALDI-MS and additional database searching constraints (number of cysteine residues in every single peptide peak) to increase the confidence of protein identification by peptide mass mapping.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of the priority of U.S.Provisional Patent Application No. 60/242,645, filed Oct. 23, 2000.

BACKGROUND OF THE INVENTION

[0002] The present invention relates to the field of high-throughputprotein analysis. More specifically, this invention relates to novelreagents for use in the identification and quantitation of proteinsusing matrix-assisted laser desorption/ionization mass spectrometry(MALDI-MS) in combination with peptide mass fingerprinting or fragmention-based database searching.

[0003] MALDI-MS has become an established tool for the rapididentification of isolated proteins and has been used inter alia toidentify proteins involved with human cancers, to elucidate componentsof multi-protein complexes, as well as for large-scale identification ofproteins in organisms with fully sequenced genomes. Prior to MALDI-MSanalysis, proteins/peptides are first separated by one-dimensional ortwo-dimensional polyacrylamide gel electrophoresis (1D or 2D-PAGE) ormultidimensional liquid chromatography. Proteins/peptides are thenidentified by peptide mass mapping or fragment ion based databasesearching. Analytical procedures involving MALDI-MS are very robust,easy to automate, and most importantly, very fast both in terms of dataacquisition and analysis.

[0004] However, peptide mass mapping may not routinely yield unambiguousprotein identification with high confidence levels, particularly whenonly a few peptides are encountered. Furthermore, MALDI-MS often yieldslower sequence coverage of proteins analyzed than electrosprayionization mass spectrometry (ESI-MS). This lower sequence coverageprimarily results from both poor recovery of hydrophobic peptides duringsample preparation and inefficient ionization of peptides withoutarginine residues by MALDI. In addition, MALDI-MS is intrinsically pooras a quantitation tool. Thus, it is very difficult to measure therelative abundance of proteins directly using MALDI-MS data.

[0005] There is a need in the art for additional reagents and methodsfor improving performance of MALDI-MS analysis of proteins/peptides bothin terms of confident identification and accurate quantitation.

SUMMARY OF THE INVENTION

[0006] In one aspect, the invention provides a method for accuraterelative quantitation of proteins using MALDI-MS. This method involvesthe following steps: reducing the disulfide bonds of proteins from abiological mixture; reacting the samples to be compared with a compoundcontaining a guanidino group attached to a thiol reactive group via alinker which can be differentially labeled with either heavy or lightisotopes (optionally prior to or following reduction); separating theproteins from the mixture; digesting the proteins; and subjecting themto quantitative mass spectrometric analysis. The compounds of theinvention are also well suited for enhancing ionization efficiency ofcysteine-containing peptides by MALDI-MS. This method is performed usingthe steps described above, with the following additional steps. Thefirst sample is labeled with a reagent with isotopic substitutions and asecond sample (e.g., a reference) is labeled with the equivalent reagentlacking these isotopic substitutions. Thereafter, the samples, oraliquots thereof, are mixed, prior to the separation step. After mixing,the modified proteins may be separated by 1 D- or 2D-PAGE, gel bands orspots are cut and subjected to in-gel enzymatic digestion andsubsequently MALDI-MS analysis. Alternatively, the mixed modifiedproteins may be subjected to enzymatic digestion and then the resultedpeptides separated by various chromatographic steps before beingsubjected to MALDI-MS analysis. The proteins may be identified bypeptide mass mapping or fragment-ion based data analysis and therelative protein abundance may be obtained by analyzing the relativepeak intensity or peak area of the same peptide from two differentsamples.

[0007] In another aspect, the invention also provides a method foraccurate relative quantitation and identification of proteins analyzedby electrospray MS. This method is performed in a manner similar to themethod described above.

[0008] In yet another aspect, the invention provides novel reagents andreagent kits containing the compounds of the invention.

[0009] Other aspects and advantages of the present invention aredescribed further in the following detailed description of the preferredembodiments thereof

DETAILED DESCRIPTION OF THE INVENTION

[0010] The inventors have identified a number of problems that cause theambiguous results in both identification and relative quantitation ofproteins using conventional approaches involving gel electrophoresis,MALDI-MS, and peptide mass mapping. More specifically, the inventorshave found that the conventional 2D-PAGE/MALDI-MS/peptide mass mappingapproach often provides inaccurate quantitation of proteins by gel imageanalysis and often ambiguous protein identifications. The inventorsbelieve that poor ionization of certain peptides by MALDI is one of themain causes for ambiguous protein identifications. The present inventionprovides reagents and methods which overcome the defects in conventionalMALDI-MS and peptide mass mapping methods.

[0011] Advantageously, the reagents of the invention can be used ascysteine-alkylating reagents, which provide many more peptide peaks inMALDI-MS than reagents previously described for use in MALDI-MS. Thisincrease in peptide peaks observed when utilizing the reagents of theinvention is due to the increased hydrophilicity and better ionizationefficiency provided to the cysteine-containing peptides. Furthermore,because the methods of the invention utilize a mixture of light andheavy reagents, the exact number of cysteine residues in all peptidepeaks observed by MALDI-MS can be determined. The resulting higherprotein sequence coverage (more peptides observed) together with theknowledge of the exact number of cysteine residues in all peptidesobserved greatly increases the specificity of database searching usingpeptide mass fingerprinting. In fact, this additional information canmake peptide mass fingerprinting routinely yield confident proteinidentifications and therefore makes the MALDI-MS combined with peptidemass fingerprinting a true high-throughput and yet unambiguous proteinidentification tool. The higher protein sequence coverage also permits amore complete chemical modification map of proteins to be obtained.Furthermore, the differential labeling strategy improves the currentlypopular 2D-PAGE-MS or 2D-LC-MS approaches in proteomics by increasingthe dynamic range and accurately quantifying individual proteins. Thisimprovement provides a much more complete picture of any proteomethrough the use of 2D-PAGE/2D-LC and MALDI-MS, thus increasing thepossibility of finding protein drug targets that are differentiallyexpressed in disease states.

[0012] Thus, the reagents of the invention are advantageous overconventional reagents for MALDI-MS analysis of proteins. These reagentscan also be used for a variety of other purposes. These reagents anduses therefore are described in more detail below.

[0013] Compounds With Guanidino Functional Groups

[0014] Cysteine-containing peptides are often more hydrophobic due tothe fact that disulfide bonds are usually buried inside of the globularproteins. Advantageously, the novel cysteine-modifying reagents of theinvention not only increase the hydrophilicity of cysteine-containingpeptides and thus minimize the loss of these hydrophobicpeptides/proteins but also, more importantly, increase the ionizationefficiency of these peptides by attachment of a guanidino functionalgroup. Although not limited to such a use, these compounds areparticularly well suited for use in MALDI-MS analysis.

[0015] In one embodiment, the compounds of the invention (ICIER) has aformula of A1-Linker-A2 which comprises a reactive group (A1) attachedto an ionization enhancement group (A2) via a linker which can bedifferentially labeled with stable isotopes (Linker). Suitably, theionization enhancement group is a strong basic functionality. In oneembodiment, the ionization enhancement group (A2) is a guanidino groupand has the formula: —NH—C(NH)—NH₂.

[0016] The linker is any structure which may be differentially labeledwith stable isotopes for use in quantitation and identification ofproteins using MALDI-MS. In one embodiment, the linker contain from 1 to100 atoms in length, about 3 to about 50 atoms in length, or about 5 toabout 15 atoms in length, which are composed of carbon, and optionally,one or two atoms selected from O, S, NH, NR, NR′, CO, C(O)O, C(O)S, S—S,SO₂, C(O)—NR′, CS—NR′, or Si—O. Optionally, one or more of the C atomsmay be substituted with a small alkyl (C₁-C₆), alkenyl, alkoxy, aryl, ordiaryl groups. For example, the linker may be an alkyl, alkenyl, oralkynyl group, optionally substituted as described above. In anotherexample, the linker may itself contain one or more O, S, NH, NR, NR′,CO, C(O)O, C(O)S, S—S, SO₂, C(O)—NR′, CS—NR′, Si—O groups bound to oneor more C atoms, which may be optionally substituted. In one embodiment,the linker is an alkyl group which contains a substitution of about fourto about twelve atoms with a stable isotope. However, the linker maycontain more than six isotope substitutions where desirable. Forexample, for peptides at the higher end of the molecular weight range atwhich MS is useful (e.g., about 2000 Da to 3500 Da) it may be desirablefor the linker to contain eight, ten, twelve or more substitutions, inorder to achieve the differential analysis required; whereas peptides atthe lower end of the molecular weight range for MS (e.g., about 500 to2000 Da) may require only four to six substitutions. For the selectednumber of substitutions, any one or more of the hydrogen, nitrogen,oxygen, carbon, or sulfur atoms in the linker may be replaced with theirisotopically stable isotopes: ²H, ¹³C, ¹⁵N, ¹⁷O, ¹⁸O, or ³⁴S.

[0017] The reactive group A1 reacts, preferably specifically, withthiols, and more particularly, with cysteine residues. Desirably, thethiol-reactive group is selected from the group consisting iodide,maleimide (see, for example, the structures below)

[0018] or α-haloacetyl groups such as X—CH₂CO—. Most suitably, the X isselected from halogens such as iodine, bromine, and chorine to formiodoacetyl, bromoacetyl, or chloroacetyl functionalities.

[0019] In another alternative, the thiol-reactive group may be selectedfrom other α-, β-conjugated double bond structures, such as

[0020] and the like. Still other reactive groups can be readilysynthesized to contain other thiol-specific reactive groups for use inbinding cysteine-containing peptides.

[0021] In certain preferred embodiments, a compound of the invention(ICIER) comprises a thiol-reactive group attached to a guanidino groupby a linker, in which the formula of the compound is:

[0022] While Compound C′ (one example of heavy ICIER) represents oneparticularly desirable isotopically heavy substituted version ofCompound C (one example of Light ICIER), other isotopically heavyversions of this formula may be readily produced according to thepresent invention. Similarly, a variety of substitutions to Compounds A,B and D may be readily generated by one of skill in the art based on theteachings provided herein.

[0023] Synthesis of Reagents

[0024] The compounds of the invention may be readily synthesized by oneof ordinary skill in the art utilizing the methods described in theexamples below and techniques known to those of skill in the art. Someexemplary methods are illustrated in Example 1 below.

[0025] For example, a suitable starting material may be mixed withL-arginine in a mixture of tetrahydrofuran (THF) and water in a ratio ofabout 1 to about 1 parts by volume, for about 10 to about 48 hours, andmost preferably about 16 hours at room temperature. The reaction mixtureis then poured into acetone and the solid is collected. The solid isthen dissolved in water and introduced into a suitable column, which iseluted with water to provide a compound of the invention. However, theinvention is not so limited. For example, other suitable solvents may besubstituted for the THF or acetone. Alternatively, the ratio of THF towater may be adjusted, as needed or desired. As another example, a saltof L-arginine (e.g., L-arginine D7-hydrochloride or L-argininamidedihydrochloride) may be dissolved in water and the pH adjusted to thebasic range (e.g., about 8 to about 13, and more preferably about 8 toabout 10), prior to reaction with iodoacetyl anhydride. Thereafter, thesolid may be collected, e.g., by lowering the pH to the acid range(e.g., about 2 to about 4), filtering the resin and extracting theaqueous solution, followed by further filtration. The resulting solidmay be freeze-dried to yield the desired compound.

[0026] However, given the descriptions provided herein, one of skill inthe art will be able to readily select appropriate techniques andreagents for synthesis of compounds of the invention.

[0027] Following synthesis, the compounds are preferably purified toachieve the best results, particularly when they will be used inconjunction with 2D-PAGE, since reagents made in situ contain an excessof salt that will interfere with the first separation step ofisoelectric focusing. Suitably, purification may be performed byfiltration. Alternatively, other suitable methods may be readilyselected by one of skill in the art.

[0028] These compounds may be utilized in a variety of methods in whichprotein/peptide labeling and/or increasing the ionization ofcysteine-containing peptides is desired. However, the compounds areparticularly useful in methods for high-throughput proteinidentification and quantitation using MALDI-MS.

[0029] Methods of Using the Compounds of the Invention

[0030] The compounds of the invention are particularly useful in methodsfor quantitation and identification of one or more proteins in amixture. Suitably, the peptides analyzed by the method of the inventionare between about 500 Daltons (Da) to about 3500 Daltons. The proteinmixture may be a sample from a cell or tissue culture, or biologicalfluids, cells or tissues. Samples from a culture include cellhomogenates and cell fractions. Biological fluids include urine, blood(including, e.g., whole blood, plasma and sera), cerebrospinal fluid,tears, feces, saliva, and lavage fluids. The mixtures may includeproteins, lipids, carbohydrates, and nucleic acids. The methods of theinvention employ MS and (MS)^(n) methods. Currently, matrix assistedlaser desorption ionization MS (MALDI/MS) and electrospray ionization MS(ESI/MS) methods are preferred. However, a variety of other MS and(MS)^(n) techniques may be selected.

[0031] In one embodiment, the invention provides a method forquantitative analysis of a proteome (i.e., a complex mixture containingproteins and/or peptides) using the compound of the invention.Typically, a sample is obtained from a source, as defined above. Whereisolated proteins will be identified using techniques based on MALDI-MSand peptide mass mapping, the sample may be compared to a referenceprotein mixture, which is obtained as a sample from the same source ormay be obtained from another source. Alternatively, isolated proteinsmay be identified using post-source delay (PSD) or collision-induceddissociation (CID) techniques followed by fragment ion-based databasesearching (M. Mann and M. Wilm, Anal. Chem., 66: 4390 (1994)) or de novosequencing, and the sample may be compared to a reference proteinmixture using MS data. The sample protein mixture and the referenceprotein mixture are processed separately, applying identical reactionconditions, with the exception that only one mixture (e.g., the sample)will be reacted with the compound containing isotopically stableisotopes. Alternatively, where relative quantitation of proteins is notdesirable, no reference samples are required; nor are isotopically heavyequivalents of the compounds of the invention required. Optionally, anylabeling reaction step may be performed prior to, or following, theother method steps which are described herein.

[0032] Typically, the protein sample is dissolved in a buffer suitablefor 1D-PAGE or 2D-PAGE or in-solution enzymatic digestion. Such buffersmay be purchased commercially from a variety of sources (e.g., GenomicSolutions, Ann Arbor, Mich.; BioRad, Hercules, Calif.) or preparedaccording to known methods. Throughout the following method steps, thepH of the mixture is maintained under neutral or basic conditions. Mostsuitably, the pH is maintained between 7 and 10. Preferably, the methodof the invention is performed at a basic pH where the compound of theinvention containing the guanidino group (e.g., compounds A, B, C, C′and D) is utilized. Most suitably, the pH is in the range of about 8 toabout 9, and most preferably about 8.5. Alternatively, the method of theinvention is preferably performed at a neutral pH where a compound ofthe invention containing a maleimide affinity tag is utilized. In thiscircumstance, the method is preferably performed at a pH of about 6.5 toabout 8.5, more preferably 7 to 8, and most preferably 7 to 7.5.

[0033] Following preparation of the sample and reference, the disulfidebonds of the proteins in the sample(s) or reference mixtures are reducedto free SH groups. Suitable reducing agents includetri-n-butylphosphine, mercaptoethylamine, dithiothreitol (DTT), andtricarboxyethylphosphine, which are used in excess. However, othersuitable reducing agents may be substituted. In one embodiment,disulfide bonds are denatured using 50 mM Tris buffer, 6M guanidine HCl,5 mM tributyl phosphine at pH 8.5 for 1 hour at 37° C. However, otherreducing agents, buffered to a pH in the basic range may be selected andincubated for varying lengths of times at room temperature.

[0034] Where no protein quantitation is to be performed, no referencesample need be labeled, and the following parallel reaction steps withequivalent heavy or light ICIER and mixing steps can be eliminated.Where protein quantitation will be performed, a selected compound of theinvention, either an isotopically heavy or light compound, will bereacted with the samples to be compared. This labeling reaction step maybe performed prior to, or following, the other method steps which aredescribed herein. Typically, the reference sample is labeled with theisotopically heavy compound and the experimental sample(s) are labeledwith the isotopically light form of the compound. However, the labelingmay be reversed. Following reduction and reaction with the selectedlabeling reagents (heavy or light ICIER), defined aliquots of thesamples (optionally labeled with isotopically different compounds, e.g.,corresponding light and heavy compounds) are combined and all thesubsequent steps are performed on the pooled samples. Preferably, equalamounts of the samples are combined.

[0035] Suitably, prepared gels for one-dimensional (1D) ortwo-dimensional (2D) polyacrylamide gel electrophoresis (PAGE) may beobtained from a variety of commercial sources and used according tomanufacturer's instruction (Genomics Solutions; Ann Arbor, Mich.; NOVEX,San Diego, Calif.). However, the invention is not so limited. One ofskill in the art can readily apply other techniques for separating theICIER-labeled proteins.

[0036] Following mixing of the ICIER-treated samples, the proteins areseparated by 1D-PAGE or 2D-PAGE. Then the protein bands or spots ofinterest are cut and subjected to enzymatic digestion. Suitably, theproteins may be subjected to in-gel digestion using techniques whichhave been described previously (e.g., Rosenfeld et al, Anal. Biochem.,203:173-179 (1992) and Sechi et al, Anal. Chem., 70:5150-5158 (1998)),or the modification thereof as described in the examples below.

[0037] A suitable protease for use in this enzymatic digestion methodmay be readily selected from among proteases which are compatible withthe basic conditions and the procedure. In one embodiment, the proteaseis trypsin. In another embodiment, a mixture of proteases which havesimilar activity levels at basic pH is used. Such proteases may includeaminopeptidases, carboxypeptides, among others. Alternatively, proteindigestion may be omitted where the proteins to be analyzed are small(e.g., about 500 to 1000 Da).

[0038] Suitably, the peptides are extracted from the gel usingconventional techniques. For example, following destaining, the peptidesmay be extracted by adding a solution of acetonitrile andtrifluoroacetic acid (TFA) to the gel band and incubating, beforecollecting the liquid phase. This step may be repeated and additionalacetonitrile added to complete the extraction. The extract solutions arepooled and dried, then reconstituted with a solution of acetonitrile andTFA. Other suitable methods for peptide extraction are well known tothose of skill in the art and may be readily utilized.

[0039] The isolated, derivatized peptides are then analyzed using MStechniques. Both the relative quantity and sequence identity of theproteins from which the labeled peptides originated can be determined byMALDI-MS techniques (i.e. MS and MS^(n) (PSD, CID)) and subsequent dataanalysis (i.e. peptide mass mapping or fragment-ion based dataanalysis). Preferably, the relative quantitation of proteins is obtainedfrom MS data, while the protein identification can derived from theanalysis of either MS data (peptide mass mapping) or MS^(n) data(fragment ion based database searching).

[0040] Apparatuses for performing MALDI-MS, and techniques for theiruse, are described in International Publication WO 93/24835, U.S. Pat.No. 5,288,644, R. Beavis and B. Chait, Proc. Natl. Acad. Sci. USA,87:6873-6877 (1990); B. Chait and K. Standing, Int. J. Mass Spectrom,Ion Phys., 40:185 (1981) and Mamyrin et al, Sov. Phys. JETP, 37:45(1973), all of which are incorporated by reference herein. Briefly, thefrequency tripled output of, e.g., a Q-switched Lumonics HY400neodymium/yttrium aluminum garnet lawer (“Nd-YAG”) (355 nm, 10-nsecoutput pulse) is focused by a lens (12-inch focal length) through afused silica window onto a sample inside the mass spectrometer. Theproduct ions formed by the laser are accelerated by a static electricpotential of 30 kV. The ions then drift down a 2-m tube maintained at avacuum of 30 μPa and their arrival at the end of the tube is detectedand recorded using, e.g., a Lecroy TR8828D transient recorder. Thetransient records of up to 200 individual laser shots are summedtogether and the resulting histogram is plotted as a mass spectrum. Peakcentroid determinations and data reduction can be performed using a VAXworkstation or other computer system.

[0041] However, other MS techniques, including electrospray ionization(ESI)/MS, among others, may be readily utilized to analyze the proteinsand peptides modified by the compounds of the invention (ICIER).

[0042] Reagent Kit

[0043] The invention further provides a reagent kit for the analysis ofproteins by mass spectral analysis. Typically, such a kit will containone or more compounds of the invention. Most suitably, the kit willcontain a set of substantially identical, differentially labeled(isotopically light and heavy) compounds. The kit may further containone or more proteolytic enzymes, reaction buffers, or wash solutions.

[0044] The method and kit of the invention may be used for a variety ofclinical and diagnostic assays, in which the presence, absence,deficiency or excess of a protein is associated with a normal or diseasestate. The method and kit of the invention can be used for qualitativeand quantitative analysis of protein expression in cells and tissues.The method and kit can also be used to screen for proteins whoseexpression levels in cells or biological fluids is affected by a drug,toxin, environmental change, or by a change in condition or cell state,e.g., disease state, malignancy, site-directed mutation, gene therapy,or gene knockouts.

[0045] The following examples are provided to illustrate the inventionand do not limit the scope thereof. One skilled in the art willappreciate that although specific reagents and conditions are outlinedin the following examples, modifications can be made which are meant tobe encompassed by the spirit and scope of the invention.

EXAMPLE 1 Reagents of the Invention

[0046] This example illustrates methods for synthesis of exemplarycompounds A, B, C, C′ and D of the invention. These compounds are usefulas reagents in MALDI-MS and peptide mass mapping, as shown in thefollowing examples.

[0047] 1.2-(2-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-acetylamino)-5-guanidino-pentanoicAcid or Maleimidoacetyl Arginine (A):

[0048] (2,5-Dioxo-2,5-dihydro-pyrrol-1-yl)-aceticacid-2,5-dioxo-pyrrolidin-1-yl ester (4.8 g, 19 mmol) and L-arginine(2.9, 17 mmol) was stirred in 30 mL of a mixture of tetrahydrofuran(THF) and water, THF:H₂O (1:1), for 16 hours at room temperature. Thereaction mixture was poured into acetone (1.5 L) and the solid wascollected. The solid was dissolved in H₂O (3 mL) and then introducedonto Bakerbond (Octadecyl (C18) 40 μm prep LC Packing) column, andeluted with water to give 1.1 g of product. ¹H NMR (D₂O, 300 MHZ): 6.8(s, 2H), 4.3 (H_(a), d, J=16.9 Hz, 1H), 4.2 (H_(b), d, J=16.9 Hz, 1H),4.1 (dd, J=4.9, 7.7 Hz, 1H), 3.1 (t, J=6.9 Hz, 2H), 1.8-1.5 (m, 4H).

[0049] 2.2-(3-2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-propionylamino)-5-guanidino-pentanoicAcid or Maleimidopropionyl Arginine (B):

[0050] 3-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-propionicacid-2,5-dioxo-pyrrolidin-1-yl ester (7.6 g, 28.5 mmol) and L-arginine(4.3 g, 25 mmol) was stirred in 50 mL of a mixture of THF:H₂O (1:1) for16 hr at room temperature. The reaction mixture was poured into acetone(1.5 L) and the solid was collected. The solid was dissolved in H₂O (5mL) and then introduced onto Bakerbond (Octadecyl (C18) 40 μm prep LCPacking) column, and eluted with water to give 1.5 g of product. ¹H NMR(D₂O, 300 MHZ): 6.8 (s, 2H), 4.1 (dd, J=4.7, 7.4 Hz, 1H), 3.8 (m, 2H),3.2 (t, J=6.9 Hz, 2H), 2.5 (m, 2H), 1.7-1.5 (m, 4H).

[0051] 3. N-α-(Iodoacetyl)-L-arginine (C):

[0052] L-Arginine (8.0 g, 45.6 mmol) was dissolved in deionized water(75 mL) and was reacted with iodoacetic anhydride (21.0 g, 59.3 mmol)with vigorous stirring for 15 min while the pH was maintained between 8and 9.5 with Dowex 1x2-100 (OH⁻). The pH was allowed to drop to ˜4 (5-10min) and 55% aqueous hydriodic acid was added to bring the pH to 2. Theresin was filtered and the aqueous hydriodic acid was extracted withdiethyl ether (3×250 ml). The aqueous layer was neutralized with Dowex1x2-100 (OH⁻) to pH 8-9, the resin was filtered and washed with waterand the resulting solution was freeze-dried to affordN-α-(Iodoacetyl)-L-arginine (11.9 g, 76% overall yield) as a fluffywhite powder. ¹H NMR (D₂O) δ 1.52-1.88 (m, 4H), 3.12 (t, 2H, J=6.7 Hz),3.76 (d, 1H, J=10.2 Hz), 3.76 (d, 1H, J=10.4 Hz), 4.06-4.10 (m, 1H); ¹³CNMR (D₂O) δ-2.0, 24.5, 28.8, 40.7, 55.1, 156.8, 171.5, 178.3. Molecularformula: C₁₈N₁₅N₄IO₃.

[0053] 4. N-α-(Iodoacetyl)-L-arginine-D₇ (C′):

[0054] L-arginine-D₇ hydrochloride (5.2 g, 28.73 mmol) was dissolved indeionized water (50 mL) and the pH was adjusted to 8 with Dowex 1x2-100(OH⁻). It was then reacted with iodoacetic anhydride (15.5 g, 43.78mmol) as above to afford N-α-(Iodoacetyl)-L-arginine-D₇ (5.5 g, overallyield 55%/o) as a fluffy white powder. ¹H NMR (D₂O) δ 3.74 (d, 1H,J=10.2 Hz), 3.82 (d, J=10.4 Hz). Molecular formula: C₈H₈D₇N₄IO₃.

[0055] 5. N-α-(Iodoacetyl)-L-argininamide hydrochloride (D):

[0056] L-Argininamide dihydrochloride (10.5 g, 42.6 mmol) was dissolvedin deionized water (75 mL) and the pH was adjusted to 13 with Dowex1x2-100 (OH⁻). Then it was reacted with iodoacetic anhydride (18.0 g,50.85 mmol) with vigorous stirring for 15 min while the pH wasmaintained between 8 and 9.5 with Dowex 1x2-100 (OH⁻). The pH wasallowed to drop to ˜4 (5-10 min) and 55% aqueous hydriodic acid wasadded to bring the pH to 2. The resin was filtered and the aqueoussolution was extracted by diethyl ether (3×250 ml). The aqueous layerwas filtered through a pad Dowex Retardion 11A8 (50 g) and washed withwater. The resultant solution was freeze-dried to affordN-α-(Iodoacetyl)-L-argininamide hydrochloride (11.1 g, 69%) as a fluffywhite powder. ¹H NMR (D₂O) δ 1.62-2.02 (m, 4H), 3.24 (t, 2H, J=6.6 Hz),3.79 (d, 1H, J=10.2 Hz), 3.89 (d, 1H, J=10.7 Hz), 4.27-4.31 (m, 1H); ¹³CNMR (D₂O) δ-2.7, 24.5, 28.3, 40.6, 53.6, 156.9, 172.5, 176.3. Molecularformula: C₈H₁₆N₅IO₂.HCl.

EXAMPLE 2 Comparative Example Demonstrating IonizationEnhancement-MALDI-MS Analysis of Six Separate Model Proteins afterTreatment with Either the Reagent of the Invention (Light Icier) or aConventional Reagent (IAA)

[0057] A. Reduction:

[0058] 90 μg each of the six model proteins, CTLA-4-IgGl, Interleukin-12(IL-12), α-Lactalbumin, Trypsinogen, Lysozyme, and Ribonuclease, weredissolved separately in 30 μL of the reaction buffer containing 5% SDS,20% glycerol, and 750 mM Tris-HCl (pH 8.45) to obtain solutions of 3μg/μL. To each solution was added a 200-fold excess (with regard tocysteine content per protein) of dithiothreitol (DTT). The reduction wasallowed to proceed for 30 minutes at 90° C., followed by cooling of thesolutions for 10 minutes.

[0059] B. Cysteine Modification:

[0060] Reagent C (light ICIER) was synthesized as described inExample 1. Each of the six proteins was alkylated using both reagent Cand iodoacetamide separately. The amount of the alkylating reagent(reagent C or iodoacetamide) was equivalent to a five-fold excess withregard to the amount of DTT used in step A. The alkylations were allowedto proceed for 1 hour at room temperature. Then 0.1 μg of each proteinlabeled by reagent C was mixed with 0.1 μg of the same protein butalkylated by iodoacetamide. Each of the six resultant protein solutionswas then mixed in a 1:1 ratio (volume to volume) with 2× Tricine loadingbuffer.

[0061] C. Gel Electrophoresis and Staining:

[0062] Each of the final resultant protein solutions described in Part B(each solution contains 0.2 μg of one of the six proteins) was loadedonto a 10%, 10-well Tricine mini-gel (Novex, San Diego, Calif.). Thegels were run according to the manufacturer's instructions and stainedwith Coomassie Blue G-250.

[0063] D. In-Gel Digestion:

[0064] Automated in-gel digestion of proteins was carried out using a96-well ProGest (Genomic Solutions, Ann Arbor, Mich.) with a proceduremodified from Rosenfeld el al, Anal. Biochem., 203:173-179 (1992) andSechi et al, Anal. Chem., 70:5150-5158 (1998). Briefly, gel bands werecut into 1×1 mm pieces and then destained by washing sequentially with50 μL each of the following solutions: (1) 200 mM NH₄HCO₃, (2) 50%methanol/10% acetic acid; (3) 40% ethanol/water and incubated for 10minutes for each step. The three washing steps were repeated 5 times andthen 100 μL of 10 nM NH₄HCO₃ was added and incubated for 10 minutes. Thegel pieces were then dehydrated by addition of 2×100 μL of acetonitrile.After removing the excess acetonitrile, the gels were rehydrated with 25μL of a solution containing 625 ng of trypsin in 10 mM NH₄HCO₃ andincubated at 37° C. for 10 hours. Peptides were extracted by adding 30μL of a solution of 50% acetonitrile/0.5% trifluoroacetic acid (TFA) andincubated for 10 minutes before collecting the liquid phase. This stepwas repeated one more time and then 30 μL of acetonitrile was added tocomplete the extraction. The extracted solutions were pooled togetherand dried completely with a SpeedVac (Savant, Holbrook, N.Y.). Finally,the dried peptide samples were reconstituted with 20 μL of anacetonitrile/water/TFA (50:50:1) solution.

[0065] E. MALDI Mass Spectrometry

[0066] Molecular weights of all peptides were determined by analyzingone-twentieth of the reconstituted peptide solution employing amatrix-assisted laser desorption ionization (MALDI) delayed extraction(DE) reflectron time-of-flight (TOF) instrument (Voyager DE-STR, PEBiosystems, Framiminghan, Mass.) equipped with a nitrogen laser (337 nm)in reflectron mode. Peptides were crystallized by mixing 0.8 μL of thesample solution with 0.8 PL of a matrix solution containing saturatedα-cyano-4-hydroxycinnamic acid in 0.5% TFA/50% acetonitrile/water.Spectra were externally calibrated using a mixture of known peptides.

[0067] Peak tables were generated from each spectrum and the data wereused to create the ionization enhancement and peak ratio tablespresented herein.

[0068] The following tables summarize the results of the MALDI-MSanalysis of the six protein samples after treatment with reagent C(ICIER) or iodoacetamide (IAA) and the comparison of the same. SeeTables I-XVIII. TABLE I CTLA4-alkylated with IAA 1:1 (1:1 @ 0.1 g/band)Detected Mass (Da) Theoretical Mass (Da) Height 1161.6112 1161.6302 1991N/A 1171.5491 N/A 1187.5239 1187.5441 1452 1485.7119 1485.7048 79442138.9793 2139.028  7551 2801.3180 2801.2677 1253  2817.9851* 2817.26261567

[0069] TABLE II CTLA4-IgG alkylated with Reagent C (1:1 @ 0.1 g/band)Detected Mass (Da) Theoretical Mass (Da) Height 1318.7222 1318.715310610 1328.6746 1328.6343 3035 1344.6331 1344.6292 3922 1642.78091642.7899 22134 2296.0863 2296.1131 8587 2958.3574 2958.3528 14422974.3377 2974.3477 1210

[0070] TABLE III Comparison of Data for CTLA4-IgG Height Ratio forCTLA4-IgG (Reagent C/IAA) Peptide Sequence SEQ ID NO: 5.33(K)NQVSLTCLVK(G) 1 N/A (R)AMDTGLYICK(V) 2 2.70 (R)AMDTGLYICK(V) 2 2.79(R)GIASFVCEYASPGK(A) 3 1.14 (R)TPEVTCVVVDVSHEDPVK(F) 4 1.15(R)WQQGNVFSCSVMHEALHNHYTQL(S) 5 0.77 (R)WQQGNVFSCSVMHEALHNHYTQK(S) 5

[0071] TABLE IV IL-12 alkylated with IAA (1:1 @ 0.1 g/band) DetectedMass (Da) Theoretical Mass (Da) Height N/A 907.4671 N/A 1412.59171412.5905 6566 N/A 1795.7274 N/A 1863.8916 1863.8507 6089 2206.13812206.0895  782

[0072] TABLE V IL-12 alkylated with Reagent C (1:1 @ 0.1 g/band)Detected Mass (Da) Theoretical Mass (Da) Height 1064.5402 1064.5523 24111569.7046 1569.6756 9028 1952.8747 1952.8125 5441 2020.9710 2020.93584543 2363.2748 2363.1746 1181

[0073] TABLE VI Comparison of Data for IL-12 Height Ratio for IL-12(Reagent C/IAA) Peptide Sequence SEQ ID NO: N/A (K)TSATVICR(K) 6 1.37(K)EFGDAGQYTCHK(G) 7 N/A (R)YYSSSWSEWASVPCS(−) 8 0.75(R)GSSDPQGVTCGAATLSAER(V) 9 1.51 (R)FTCWWLTTISTDLTFSVK(S) 10

[0074] TABLE VII α-Lactoalbumin alkylated with IAA (1:1 @ 0.1 g/band)Detected Mass (Da) Theoretical Mass (Da) Height none  707.3398 N/A1091.51432 1091.5196 6131 none 1715.7508 N/A 1779.83739 1779.8410 3436none 1843.8458 N/A 1892.92914 1892.9250 7606 2003.914  2003.8187 overlap2591.27206 2591.1077 3115

[0075] TABLE VIII α-Lactoalbumin alkylated with Reagent C (1:1 @ 0.1g/band) Detected Mass (Da) Theoretical Mass (Da) Height  864.42181 864.4249 6976 1248.60859 1248.6047 33460 1872.83547 1872.8359 174832093.96824 2094.0112 3307 2000.93299 2000.9309 11339 2207.103242207.0953 7629 2317.96953 2317.989  5207 none 3062.3631 N/A

[0076] TABLE IX Comparison of Data for α-Lactoalbumin Height Ratio forα-Lactoalbumin (Reagent C/IAA) Peptide Sequence SEQ ID NO: N/A(K)ALCSEK(L) aa 1-8 of SID NO:11 5.46 (K)LDQWLCEK(L) aa 7-16 of SIDNO:11 N/A (K)FLDDDLTDDIMCVK(K) aa 1-16 of SID NO:12 0.96(K)ALCSEKLDQWLCEK(L) 11 N/A (K)FLDDDLTDDIMCVKK(I) 12 1.00(K)ALCSEKLDQWLCEKL(−) 11 N/A (K)DDQNPHSSNICNISCDK(F) aa 5-23 of SIDNO:12 N/A (K)IWCKDDQNPHSSNICNISCDK(F) 13

[0077] TABLE X Lysozyme alkylated with IAA (1:1 @ 0.1 g/band) DetectedMass (Da) Theoretical Mass (Da) Height N/A 505.2557 N/A none 577.2880N/A  993.4044 993.4001 16094 1065.4958 1065.5185 5466 1325.61481325.6312 7685 1333.6643 1333.6687 25788 N/A 1491.6552 Buried 2181.04652181.0300 8876 2508.5231 2508.29788 2902 2735.4461 2735.2636 2672

[0078] TABLE XI Lysozyme alkylated with Reagent C (1:1 @ 0.1 g/band)Detected Mass (Da) Theoretical Mass (Da) Height 662.3187 662.3408 11061734.3755 734.3732 2407 1150.4807 1150.4852 9782 1222.5917 1222.6036 91991428.6595 1428.7164 56959 1490.7545 1490.7538 24700 1648.7478 1648.74038750 2338.1091 2338.1151 5656 2979.3921 2979.4450 1867 2892.48202892.3487 2113

[0079] TABLE XII Comparison of Data for Lysozyme Height Ratio forLysozyme (Reagent C/IAA) Peptide Sequence SEQ ID NO: N/A (R)GCRL(−) 14N/A (R)NRCK(G) 15 0.61 (R)WWCNDGR(T) aa 1-9 of SID NO:18 1.68(R)CELAAAMKR(H) 16 7.41 (R)GYSLGNWVCAAK(F) aa 1-14 of SID NO:21 0.96(R)CKGTDVQAWIR(G) 17 N/A (R)WWCNDGRTPGSR(N) 18 0.64(R)HGLDNYRGYSLGNWVCAAK(F) 19 0.64 (R)NLCNIPCSALLSSDITASVNCAK(K) 20 0.79(R)GYSLGNWVCAAKFESNFNTQATNR(N) 21

[0080] TABLE XIII Ribonuclease A alkylated with IAA (1:1 @ 0.1 g/band)Detected Mass (Da) Theoretical Mass (Da) Height 1504.6742 1504.6825 60412224.0861 2224.1065 22526 2517.2230 2517.2890 14079 2867.4150 2867.56664222

[0081] TABLE XIV Ribonuclease A alkylated with Reagent C (1:1 @ 0.1g/band) Detected Mass (Da) Theoretical Mass (Da) Height 1661.75931661.7785 16953 2381.1712 2381.1743 46801 2831.3933 2831.3889 93323024.5002 3024.7205 4879

[0082] TABLE XV Comparison of Data for Ribonuclease Height Ratio forRibonuclease (Reagent C/IAA) Peptide Sequence SEQ ID NO: 2.81(R)ETGSSKYPNCAYK(T) 22 2.08 (K)HIIVACEGNPYVPVHFDASV(−) aa 7-27 of SIDNo.24 0.66 (R)CKPVNTFVHESLADVQAVCSQK(N) 23 1.16(K)TTQANKHIIVACEGNPYVPVHFDASV(−) 24

[0083] TABLE XVI Trypsinogen alkylated with IAA (1:1 @ 0.1 g/band)Detected Mass (Da) Theoretical Mass (Da) Height 1168.59697 1168.58255577 none 1077.5250 N/A none 1478.7347 N/A 1490.75058 1490.7426 48911609.68206 1609.6586 1573 none 2267.0110 N/A

[0084] TABLE XVII Trypsinogen alkylated with Reagent C (1:1 @ 0.1g/band) Detected Mass (Da) Theoretical Mass (Da) Height 1325.683711325.6676 24135 1234.6102 1234.61265 12845 1792.94896 1792.9050 24551647.85269 1647.8277 14787 1923.88166 1923.8289 2795 2424.063822424.0964 3046

[0085] TABLE XVIII Comparison of Data for Trypsinogen Height Ratio forTrypsinogen (Reagent C/IAA) Peptide Sequence SEQ ID NO: 4.33(K)VCNYVSWIK(Q) 25 N/A (K)APTISDSSCK(S) 26 N/A (K)CLKAPILSDSSCK(S) 273.02 (K)LQGIVSWGSGCAQK(N) 28 1.78 (K)DSCQGDSGGPVVCSGK(L) 29 N/A(K)SAYPGQITSNMFCAGYLEGGK(D) 30

[0086] From these data, it was concluded that, compared to theconventional cysteine-alkylating reagent Iodoacetamide, ICIER greatlyincreases the ionization efficiency of cysteine-containing trypticpeptides with a lysine residue at their C-terminus. For example, in theMALDI-MS spectrum of tryptic peptides from lysozyme modified by bothreagent C and IAA (see Table XII), the intensity of a peptide modifiedby reagent C (GYSLGNWVCAAK, aa 2-13 of SEQ ID NO:21, molecular weight,1428.72 Da) is 7.4 times that of the same peptide modified by IAA(molecular weight, 1325.63 Da). Moreover, mass peaks for manycysteine-containing peptides with a lysine residue at their C-terminusthat were absent when using IAA became well observed when using ICIER.On the other hand, ICIER modification did not have any significanteffect on the ionization of peptides with an arginine residue at theirC-terminus. As a result, the overall number of cysteine-containingpeptides detected by MALDI-MS is also increased, and hence the sequencecoverage obtained for proteins being analyzed when using ICIER is muchhigher than that when using iodoacetamide.

EXAMPLE 3 Protein Quantitation by Icier and MALDI-MS Using CTLA4-IgG asa Model Protein

[0087] Different amounts of CTLA4-IgG with ratios of 1:1, 1:1.5, 1:2,1:5, and 1:10 were reduced by DTT as described in EXAMPLE 2. The samplesto be compared were alkylated with either light or heavy ICIER and thenmixed together before being subjected to gel electrophoresis, proteinstaining, in-gel digestion, and MALDI-MS analysis using the sameconditions described in EXAMPLE 2.

[0088] Table XIX summarizes the labeling of CTLA4-IgG at differentratios using light and heavy ICIER. Peptide masses were all externallycalibrated using default calibration files. The relative quantitation ofthe protein from two different pools was determined by averaging themass intensity ratios between all seven pairs of peptides labeled bylight and heavy ICIER. TABLE XIX Theor. Mass (Da) Peptide Sequence SEQID NO: Observed Ratios* 1318.72 (K)NQVSLTCLVK(G) 1 1.23 1.64 2.29  4.22 8.9 1328.63 (R)AMDTGLYICK(V) 2 0.88 0.62 1.43  0.61  0.7 1374.63(R)AMDTGLYICK(V) 2 0.95 1.39 2.01  4.31  8.2 1642.79(R)GIASFVCEYASPGK(A) 3 0.96 1.39 1.86  3.68 10 2296.11(R)TPEVTCVVVDVSHEDPEVK(F)** 4 1.02 1.52 2.3  4.62  8.6 2958.35(R)WQQGNVFSCSVMHEALHNHYTQK(S) 5 0.88 0.84 1.61  2.88  2.9 2974.35(R)WQQGNVFSCSVMHEALHNHYTQK(S) 5 0.72 1.59 2.18  5.01 11 Mean of theObserved Ratio 1.04 1.47 2.09  4.41  9.38 Expected Ratio 1 1.5 2  5 10Standard Deviation 0.04 0.1 0.19  0.56  1.2 % Error 4 1.83 4.38 11.9 6.25

[0089] From these data, it was illustrated that the observed ratiosclosely reflect the expected ratios of light to heavy ICIER labeledpeptides, especially when mass peaks that give very weak intensity oroverlap with other mass peaks were excluded. The percentage error ofquantitation using the ICIER approach is less than 12% for all ratioswhich is very accurate in contrast to densitometry. Furthermore, theICIER approach is also capable of quantifying multiple proteins in asingle sample, gel band or spot since the quantitation is based onpeptides with known sequence identity.

[0090] From these data, it was also concluded that with the use of amixture of light and heavy ICIER, an exact number of cysteine residuescontained in each detected MS peak can be determined based on thepresence or absence of the isotopically labeled pairs without extrasample manipulation (see TABLE XX). This additional information can bereadily used with peptide masses for a more constrained peptide massmapping to give confident protein identification. This is particularlyuseful when dealing with more than one protein in an analysis or only alimited number of mass peaks. TABLE XX Identification of the exactnumber of cysteine residues in each MS peaks Experimental Exact NumberSequence of Cysteine-Containing Mass (Da) of Cysteines Peptides Derivedfrom CTLA4-IgG: SEQ ID NO: 587.1578 0 951.2411 0 1286.6826 0 1318.69021—light (K)NQVSLTC*LVK(G) 1 1325.6597 1—heavy (K)NQVSLTC**LVK(G) 11344.7691 1—light (R)AM*DTGLYIC*K(V) 2 1351.6682 1—heavy(R)AM*DTGLYIC**K(V) 2 1481.7472 0 1642.8055 1—light(R)GIASFVC*EYASPGK(A) 3 1649.9140 1—heavy (R)GIASFVC**EYASPGK(A) 31677.6275 0 1689.7607 0 1807.8651 0 1872.8749 0 2296.0488 1—light(R)TPEVTC*VVVDVSHEDPEVK(F) 4 2303.1326 1—heavy(R)TPEVTC**VVVDVSHEDPEVK(F) 4 2958.8857 1—light(R)WQQGNVFSC*SVMHEALHNHYTQK(S) 5 2965.5758 1—heavy(R)WQQGNVFSC**SVMHEALHNHYTQK(S) 5 2974.3697 1—light(R)WQQGNVFSC*SVM*HEALHNHYTQK(S) 5 2981.1270 1—heavy(R)WQQGNVFSC**SVM*HEALHNHYTQK(S) 5 3336.6515 0

[0091] All publications cited in this specification are incorporated byreference herein. While the invention has been described with referenceto a particularly preferred embodiment, it will be appreciated thatmodifications can be made without departing from the spirit of theinvention. Such modifications are intended to fall within the scope ofthe appended claims.

1 30 1 12 PRT Peptide of CTLA4-IgG 1 Lys Asn Gln Val Ser Leu Thr Cys LeuVal Lys Gly 1 5 10 2 12 PRT Peptide of CTLA4-IgG 2 Arg Ala Met Asp ThrGly Leu Tyr Ile Cys Lys Val 1 5 10 3 16 PRT Peptide of CTLA4-IgG 3 ArgGly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala 1 5 10 15 421 PRT Peptide of CTLA4-IgG 4 Arg Thr Pro Glu Val Thr Cys Val Val ValAsp Val Ser His Glu Asp 1 5 10 15 Pro Glu Val Lys Phe 20 5 25 PRTPeptide of CTLA4-IgG 5 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser ValMet His Glu Ala 1 5 10 15 Leu His Asn His Tyr Thr Gln Leu Ser 20 25 6 10PRT Peptide of Interleukin (IL) - 12 6 Lys Thr Ser Ala Thr Val Ile CysArg Lys 1 5 10 7 14 PRT Peptide of IL-12 7 Lys Glu Phe Gly Asp Ala GlyGln Tyr Thr Cys His Lys Gly 1 5 10 8 16 PRT Peptide of IL-12 8 Arg TyrTyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro Cys Ser 1 5 10 15 9 21PRT Peptide of IL-12 9 Arg Gly Ser Ser Asp Pro Gln Gly Val Thr Cys GlyAla Ala Thr Leu 1 5 10 15 Ser Ala Glu Arg Val 20 10 20 PRT Peptide ofIL-12 10 Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr Asp Leu Thr Phe1 5 10 15 Ser Val Lys Ser 20 11 16 PRT Peptide of alpha-Lactoalbumin 11Lys Ala Leu Cys Ser Glu Lys Leu Asp Gln Trp Leu Cys Glu Lys Leu 1 5 1015 12 17 PRT Peptide of alpha-Lactoalbumin 12 Lys Phe Leu Asp Asp AspLeu Thr Asp Asp Ile Met Cys Val Lys Lys 1 5 10 15 Ile 13 23 PRT Peptideof alpha-Lactoalbumin 13 Lys Ile Trp Cys Lys Asp Asp Gln Asn Pro His SerSer Asn Ile Cys 1 5 10 15 Asn Ile Ser Cys Lys Asp Phe 20 14 5 PRTPeptide of Lysozyme 14 Arg Gly Cys Arg Leu 1 5 15 6 PRT Peptide ofLysozyme 15 Arg Asn Arg Cys Lys Gly 1 5 16 11 PRT Peptide of Lysozyme 16Arg Cys Glu Leu Ala Ala Ala Met Lys Arg His 1 5 10 17 13 PRT Peptide ofLysozyme 17 Arg Cys Lys Gly Thr Asp Val Gln Ala Trp Ile Arg Gly 1 5 1018 14 PRT Peptide of Lysozyme 18 Arg Trp Trp Cys Asn Asp Gly Arg Thr ProGly Ser Arg Asn 1 5 10 19 21 PRT Peptide of Lysozyme 19 Arg His Gly LeuAsp Asn Tyr Arg Gly Tyr Ser Leu Gly Asn Trp Val 1 5 10 15 Cys Ala AlaLys Phe 20 20 25 PRT Peptide of Lysozyme 20 Arg Asn Leu Cys Asn Ile ProCys Ser Ala Leu Leu Ser Ser Asp Ile 1 5 10 15 Thr Ala Ser Val Asn CysAla Lys Lys 20 25 21 26 PRT Peptide of Lysozyme 21 Arg Gly Tyr Ser LeuGly Asn Trp Val Cys Ala Ala Lys Phe Glu Ser 1 5 10 15 Asn Phe Asn ThrGln Ala Thr Asn Arg Asn 20 25 22 15 PRT Peptide of Ribonuclease 22 ArgGlu Thr Gly Ser Ser Lys Tyr Pro Asn Cys Ala Tyr Lys Thr 1 5 10 15 23 24PRT Peptide of Ribonuclease 23 Arg Cys Lys Pro Val Asn Thr Phe Val HisGlu Ser Leu Ala Asp Val 1 5 10 15 Gln Ala Val Cys Ser Gln Lys Asn 20 2427 PRT Peptide of Ribonuclease 24 Lys Thr Thr Gln Ala Asn Lys His IleIle Val Ala Cys Glu Gly Asn 1 5 10 15 Pro Tyr Val Pro Val His Phe AspAla Ser Val 20 25 25 11 PRT Peptide of Trypsinogen 25 Lys Val Cys AsnTyr Val Ser Trp Ile Lys Gln 1 5 10 26 12 PRT Peptide of Trypsinogen 26Lys Ala Pro Ile Leu Ser Asp Ser Ser Cys Lys Ser 1 5 10 27 15 PRT Peptideof Trysinogen 27 Lys Cys Leu Lys Ala Pro Ile Leu Ser Asp Ser Ser Cys LysSer 1 5 10 15 28 16 PRT Peptide of Trypsinogen 28 Lys Leu Gln Gly IleVal Ser Trp Gly Ser Gly Cys Ala Gln Lys Asn 1 5 10 15 29 18 PRT Peptideof Trypsinogen 29 Lys Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Val ValCys Ser Gly 1 5 10 15 Lys Leu 30 23 PRT Peptide of Trypsinogen 30 LysSer Ala Tyr Pro Gly Gln Ile Thr Ser Asn Met Phe Cys Ala Gly 1 5 10 15Tyr Leu Glu Gly Gly Lys Asp 20

What is claimed is:
 1. A method for enhancing identification andrelative quantitation of proteins and peptides using mass spectrometry(MS), said method comprising the steps of: (a) reducing the disulfidebonds of a first sample from a biological mixture containing proteinsand peptides; (b) labeling proteins and peptides in the first samplewith a reagent which comprises a thiol-specific reactive group attachedto a guanadino group via a linker which can be differentially labeled;(c) separating the proteins and peptides from the sample; (d) digestingthe proteins to provide a mixture containing digestion peptides andpeptides from the first sample; and (e) subjecting the peptides of (d)to quantitative MS analysis and protein identification.
 2. The methodaccording to claim 1, wherein the peptides of (d) are subjected tomatrix-assisted laser desorption/ionization (MALDI)-MS.
 3. The methodaccording to claim 1, wherein the reagent comprises a thiol-specificreactive group is selected from the group consisting of α-haloacetyl(—X—CH₂CO—, X═I, Br, or Cl) or a maleimide group having a structureselected from the group consisting of


4. The method according to claim 1, wherein the linker comprises analkyl chain having three to eight carbon atoms, optionally substitutedwith one or more amido groups, carboxy groups, or amino groups.
 5. Themethod according to claim 1, wherein the proteins and peptides arefurther subjected to peptide mass mapping, said method furthercomprising the steps of: labeling proteins and peptides in a secondsample with said reagent having heavy stable isotopes; and mixing thefirst and second samples prior to the separation step, wherein thereagent in the labeling step contains light stable isotopes.
 6. Themethod according to claim 1, wherein the linker in the reagent of step(b) contains a substitution of four to twelve atoms with a stableisotope.
 7. The method according to claim 6, wherein the linker containsseven stable isotopes.
 8. The method according to claim 6, wherein thehydrogen atoms are substituted with deuterium.
 9. The method accordingto claim 5, wherein the reagent is selected from the group consistingof:


10. The method according to claim 5, wherein the separation step isperformed using one dimensional or two dimensional polyacrylamide gelelectrophoresis (1D or 2D-PAGE), or liquid chromatography.
 11. Themethod according to claim 1, wherein the digestion step is performedin-gel or in solution.
 12. A method for preparing peptides for MALDI-MSand subsequent data analysis, said method comprising the steps of: (a)reducing the disulfide bonds of proteins from biological samples; (b)labeling proteins in one sample with a reagent which comprises athiol-specific reactive group attached to a guanidino group via a linkerwhich is differentially labeled with light stable isotopes; (c) labelingproteins in a second sample with a reagent having heavy stable isotopes;(d) mixing the first and second labeled samples; (e) separating theproteins from the mixture; (f) digesting the proteins, thereby providingpeptides ready for MALDI-MS analysis and protein identification.
 13. Themethod according to claim 11, wherein the digestion step is performedusing trypsin.
 14. A compound useful in quantitative analysis of proteinmixtures, said compound comprising a thiol-specific reactive groupattached to a guanidino group via a linker which can be differentiallylabeled with stable isotopes.
 15. The compound according to claim 14,wherein the linker contains four to twelve stable isotopes.
 16. Thecompound according to claim 14, wherein the linker contains asubstitution of at least six hydrogen atoms with deuterium.
 17. Thecompound according to claim 14, selected from the group consisting of:


18. A reagent kit for the analysis of proteins by mass spectrometricanalysis that comprises a compound of claim 14 or claim
 17. 19. Thereagent kit according to claim 18, comprising a set of substantiallyidentical differentially labeled alkylating reagents.
 20. The reagentkit according to claim 18, further comprising one or more proteolyticenzymes for use in digestion of proteins modified by said compounds.